| Antimony(Sb)is a kind of heavy metal which has a strong toxic effect on organisms,and its compounds have been listed as carcinogens by the International Agency for Research on Cancer(IARC).The production and reservation of Sb in China rank first in the World.With the development of industrial applications and exploitation of mineral resources,antimony pollution has become an increasingly serious problem.However,some microorganisms can grow and breed in the high concentration of antimony environment,and even can use antimony as an energy source,or convert Sb(?)to Sb(?),so as to reduce the toxicity of Sb in the environment.It was shown that deletion of arsenite[As(?)]oxidase gene aioA reduced Sb(?)oxidation rate by one-third in Agrobacterium tumefaciens 5A.Sequence comparison analysis found that the sequence of AioA in GW4 and 5A are 93%similar.Therefore,we poposed that AioBA may also has the ability to convert Sb(?)to Sb(?)in Agrobacterium tumefaciens GW4.In addition,the mutant strain GW4-?aioA and the complementary strain GW4-?aioA-C were obtained from previous work.The aim of this study is to clarify the influence of AioA on antimonite oxidation,chemotaxis and energy metabolism in Agrobacterium tumefacians GW4.Comprehensive analyses of the results showed that deletion of aioA has obvious effect on the growth and Sb(?)resistance of strain GW4,however,the Sb(?)oxidation rate was slightly increased in the presence of 50 ?M Sb(?).Moreover,the mutant strain GW4-?aioA displayed a stronger positive chemotaxis ability to Sb(?)compared with the wild type strain GW4.To investigate the effects of AioA on Sb(?)oxidation and chemotaxis,dimensional electrophoresis was employed and 21 proteins were successfully identified by MS-MS.Differential expression proteins were classified according to the KEGG metabolism pathway analysis(metabolism 59%,genetic information processing 3%,environmental information processing 20%,cellular processes 3%and unspecified 15%).Then,qRT-PCR,HPLC and microcalorimetry were employed in further studies.The results indicated that:(1)The expression of Sb(?)oxidase AnoA and H2O2-mediated non-enzymaticbacterial Sb(?)oxidation were both increased by deletion of aioA.Thus,the Sb(?)oxidation rate was increased in mutant strain GW4-?aioA.(2)The amino acid metabolism in GW4-?aioA was enhanced compared with the wild type strain.(3)The carbohydrate metabolismin strain GW4 was induced by Sb(?),and deletion of aioA resulted in a further enhancement.(4)The ATP and NADH contents of GW4-?aioA were higher than GW4 after 2 h of incubation with 50 ?M Sb(?).(5)Deletion of aioA increased the heat production of strain GW4 with the addition of 50 ?M Sb(?).The energy generated from TCA cycle and Sb(?)oxidation may be used for the detoxication of Sb(?),and finally released in the form of heat.In all of the experiments,the phenotype of the complementary strain GW4-?aioA-C was basically recovered.This study demonstrated that the arsenic oxidase gene aioA could influence the bacterial antimonite oxidation,chemotaxis and energy metabolism in A.tumefaciens GW4.Bacterial Sb(?)oxidation catalyzed by AnoA and H2O2 were both increased in GW4-?aioA,which may related to the change of the regulatoty mechanisms affected by aioA.In addition,the regulatry factors involved in bacterial chemotaxis may also be influenced by aioA.The TCA cycle in GW4-?aioA was increased along with the increasing Sb(?)oxidation and chemotaxis,which produced more ATP and NADH for antimony resistance.Ultimately,the energy generated from TCA cycle and Sb(?)oxidation was partially released in the form of heat.The results of this thesis explained a novel detoxification mechanism for bacterial Sb(?)oxidation which has a significant scientific impact. |
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